Intracuticular lipids of spinach leaves

The cuticular wax and cutin components of the cuticular membranes isolated from the leaves of two spinach cultivars have been determined. The membranes contain about 0·007 mg/cm² of cuticular wax which comprises monobasic acids (C₁₆–C₃₈) with hexadecanoic as the major component. The amounts of cutin are comparable with those of cuticular wax and the monomeric constituents are predominantly C₁₈ epoxy compounds. The most abundant monomer is 9,10-epoxy-18-hydroxyoctadecanoic acid (up to 63%) together with substantial amounts of 9,10,18-trihydroxyoctadecanoic acid (up to 22%). Also present are 9,10-epoxyoctadecane-1,18-dioic acid (6–7%) dihydroxyhexadecanoic acid (3–4%) and ω-hydroxymonobasic and fatty acid fractions. The tentative identification of two minor components, 18-hydroxyoxooctadecanoic and 9,10-epoxy-12,18-dihydroxyoctadecanoic acids, is also made. Although spinach membranes have a delicate structure their cutin composition is essentially similar to that of much more substantial membranes.     

Reduction of the Acyl Group: The Critical Step in Bombykol Biosynthesis That Is Regulated in Vitro by the Neuropeptide Hormone in the Pheromone Gland of Bombyx mori

The sex pheromone of Bombyx mori, bombykol [(10E,12Z)-10,12-hexadecadien-1-ol], can be biosynthesized in four steps: construction of a hexadecanoic moiety from acetyl CoA, ?-11-desaturation, .?-10,12-desaturation, and reduction of the acyl group. This biosynthesis is regulated by a hormone named the pheromone biosynthesis activating neuropeptide (PBAN). To examine the steps that are accelerated by this neurohormone, pheromone glands excised from decapitated females were incubated in vitro with either ¹⁴C-Iabeled sodium acetate or one of three fatty acids [hexadecanoic acid, (Z)-11-hexadecenoic acid, or (10E,12Z)-10,12-hexadecadienoic acid]. After analyzing the radioactivity that was incorporated from each precursor into bombykol and the biosynthetic precursors, it was observed that the first three steps proceeded in glands both treated and untreated with synthetic PBAN of B. mori; however, the last step proceeded only in the treated glands. From this in vitro experiment, it can be concluded that the main regulatory role of PBAN is in the reduction of the acyl group in B. mori, as was shown by our previous in vivo experiment.     

Identification of a novel fatty acid elongase with a wide substrate specificity from arachidonic acid-producing fungus <Emphasis Type="Italic">Mortierella alpina</Emphasis> 1S-4

The isolation and characterization of a gene (MALCE1) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4 are described. MALCE1 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae, resulting in the accumulation of 18-, 19-, and 20-carbon monounsaturated fatty acids and eicosanoic acid. Furthermore, the MALCE1 yeast transformant efficiently elongated exogenous 9-hexadecenoic acid, 9,12-octadecadienoic acid, and 9,12,15-octadecatrienoic acid. The MALCE1 gene-silenced strain obtained from M. alpina 1S-4 exhibited a low content of octadecanoic acid and a high content of hexadecanoic acid, compared with those in the wild strain. The enzyme encoded by MALCE1 was demonstrated to be involved in the conversion of hexadecanoic acid to octadecanoic acid, its main role in M. alpina 1S-4.     

Hormonal effects on fatty acid binding and physical properties of rat liver plasma membranes

Summary The fluorescent fatty acids,trans-parimaric andcis-parinaric acid, were used as analogs of saturated and unsaturated fatty acids in order to evaluate binding of fatty acids to liver plasma membranes isolated from normal fed rats. Insulin (10^–8 to 10^–6 m) decreasedtrans-parinaric acid binding 7 to 26% whilecis-parinaric acid binding was unaffected. Glucagon (10^–6 m) increasedtrans-parinaric acid binding 44%. The fluorescence polarization oftrans-parinarate,cis-parinarate and 1,6-diphenyl-1,3,5-hexatriene was used to investigate effects of triiodothyronine, insulin and glucagon on the structure of liver plasma membranes from normal fed rats or from rats treated with triiodothyronine or propylthiouracil. The fluorescence polarization oftrans-parinarate,cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene was 0.300±0.004, 0.251±0.003, and 0.302±0.003, respectively, in liver plasma membranes from control rats and 0.316±0.003, 0.276±0.003 and 0.316±0.003, respectively, in liver plasma membranes from hyperthyroid rats (p<0.025,n=5). Propylthiouracil treatment did not significantly alter the fluorescence polarization of these probe molecules in the liver plasma membranes. Thus, liver plasma membranes from hyperthyroid animals appear to be more rigid than those of control animals. The effects of triiodothyronine, insulin and glucagon addedin vitro to isolated liver plasma membrane preparations were also evaluated as follows: insulin (10^–10 m) and triiodothyronine (10^–10 m) increased fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in liver plasma membranes while glucagon (10^–10 m) had no effects. These hormonal effects on probe fluorescence polarization in liver plasma membranes were abolished by pretreatment of the rats for 7 days with triiodothyronine. Administration of triiodothyronine (10^–10 m)in vitro increased the fluorescence polarization of trans-parinaric acid in liver plasma membranes from propylthiouracil-treated rats. Thus, hyperthyroidism appeared to abolish thein vitro increase in polarization of probe molecules in the liver plasma membranes. Temperature dependencies in Arrhenius plots of absorption-corrected fluorescence and fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene were noted near 25°C in liver plasma membranes from triiodothyronine-treated rats and near 18°C in liver plasma membranes from propylthiouracil-treated rats. In summary, hormones such as triiodothyronine, insulin and glucagon may at least in part exert their biological effects on metabolism by altering the structure of the liver plasma membranes.     

The constituent cutin acids of cranbury cuticle

Purified cutin from cranberry (Vaccinium macrocarpon, var. Howes) skin was selectively degraded, and the cutin acids, as methyl esters, separated by TLC into seven classes including monobasic acids, dibasic acids, monohydroxy monobasic acids, monohydroxy epoxymonobasic acids, vic-dihydroxy dibasic acids, dihydroxy monobasic acids and trihydroxy monobasic acids. Of the 41 components identified in cranberry cutin by GLC and MS analysis, 18-hydroxyoctadec-cis-9-enoic acid (9·4%), 18-hydroxy-cis-9,10-epoxyoctadecanoic acid (7·5%), 10,16-dihydroxyhexadecanoic acid (16·7%) and threo-9,10,18-trihydroxyoctadecanoic acid (43·7%) were shown to be the major constituents.     

Effects of Growth Regulators on H+Translocation across the Membranes of Plasma Membrane Vesicles from Potato Tuber Cells

Effects of the growth regulators epibrassinolide-694 (EB), gibberellic acid (GA), and abscisic acid (ABA) on the ATP-dependent translocation of H⁺through the membranes of plasma membrane vesicles of potato (Solanum tuberosumL.) tuber cells were studied. The ATP-dependent accumulation of H⁺in the plasma membrane vesicles from dormant tubers was inhibited by EB and ABA and stimulated by GA. After the break of dormancy, the stimulatory effect of GA increased, the inhibitory effect of ABA decreased, and EB stimulated the accumulation of H⁺in the vesicles. The data suggest that the plasma membrane H⁺ATPase is a target of phytohormones that regulate the dormancy of potato tubers.     

Synthesis of ethyl-(3R,5S)-dihydroxy-6-benzyloxyhexanoates via diastereo- and enantioselective microbial reduction: Cloning and expression of ketoreductase III from Acinetobacter sp. SC 13874

Previously we have demonstrated the reduction of ethyl and t-butyl diketoesters 1 to the corresponding syn-(3R,5S)-dihydroxy esters 2a by Acinetobacter sp. 13874. The syn-(3R,5S)-dihydroxy ester 2a was obtained with an enantiomeric excess (e.e.) of 99% and a diastereomeric excess (de) of 63%. In this report, we identified a gene encoding desired ketoreductase III which catalyzed the diastereoselective reduction of diketoesters 1 to syn-(3R,5S)-dihydroxy esters 2a and describe cloning and expression of ketoreductase III into Escherichia coli. Cells or extracts of recombinant E. coli efficiently reduced the diketoester 1 to the corresponding syn-(3R,5S)-dihydroxy ester 2a in 99.3% yield, 100% e.e., and 99.8% de.     

Effects of fatty acids on activity and calmodulin binding of Ca-ATPase of human erythrocyte membranes

Activation and inhibition of Ca²⁺-ATPase of calmodulin-depleted human erythrocyte membranes by oleic acid and a variety of other fatty acids have been measured. Low concentrations of oleic acid stimulate the enzyme activity, both in the presence and in the absence of calmodulin. Concomitantly, the affinity of the membrane bound enzyme to calmodulin progressively decreases due to competitive interactions of calmodulin and oleic acid with the enzyme. Removal of oleic acid from the membrane by serum albumin extinguishes the activating effect of oleic acid and restores the ability of the enzyme to bind calmodulin with high affinity. High concentrations of oleic acid induce an almost complete and irreversible loss of enzyme activity which cannot be abolished by removal of oleic acid. Despite a complete loss of enzyme activity, binding of calmodulin to membranes is approximately normal after removal of oleic acid. Activities of (Na⁺ + K⁺)-ATPase, Mg²⁺-ATPase and acetylcholine esterase, as well as the total protein content, show no gross changes upon treatment of membranes with increasing amounts of oleic acid, which seems to exclude that membrane solubilisation by oleic acid causes an inactivation of the enzyme.     

Phenotypic and molecular identification of Cellulosimicrobium cellulans isolated from Antarctic snow

We report for the first time the isolation of Cellulosimicrobium cellulans from Antarctic snow. This strain demonstrated physiological traits that were markedly different from that of the mesophilic C. cellulans type strain DSM 43879^T. The dominant cell wall sugars in C. cellulans were glucose, galactose and mannitol whereas rhamnose was the only major sugar in the type strain. Cellular fatty acid patterns were dominated by 12-methyltetradecanoic acid (ai-C_15:0), hexadecanoic acid (C_16:0) and 14-methylhexadecanoic acid (ai-C_17:0) but lacked iso fatty acids unlike the type strain. The ability of C. cellulans to survive in Antarctic snow could be due to these modified physiological properties that distinguish it from its mesophilic counterpart. Carbon utilization studies demonstrated that C. cellulans preferred complex carbon substrates over simple ones suggesting that it could play a potential role in carbon uptake in snow. Our study shows that this genus could be more cosmopolitan than hitherto thought of and is capable of living in extreme cold environments.     

Effect of Inoculation with Various Rhizobia Strains on the Fatty Acid Composition of Peribacteroid and Bacteroid Membranes of the Nodule Symbiosomes

The fatty acid (FA) composition of bacteroid and peribacteroid membranes was studied in the symbiotic pairs differing in their nitrogen-fixing efficiency; the results are compared with the FA composition of plasmalemma and free-living rhizobia. The experiments involved lupine plants inoculated with strains of Bradyrhizobium lupini359a (Nod⁺Fix⁺) and 400 (Nod⁺Fix L) manifesting high and low nitrogen-fixing efficiency, respectively, and broad bean plants inoculated with strains of Rhizobium leguminosarum97 (Nod⁺Fix⁺) and 87 (Nod⁺Fix L) of high and low nitrogen-fixing efficiency, respectively. We showed that the rhizobia of the strains 359a and 97 were able to form nodules with peribacteroid membranes containing FA mainly or exclusively of plant origin. These strains were able to develop effective symbiotic pairs with legume plants. The use of strains 400 and 87 resulted in the formation of nodules with peribacteroid membranes containing typical bacterial (branched-chain) FAs; these strains were characterized by an ineffective symbiosis.     

Continous replication of potato spindle tuber viroid (PSTV) in permanent cell cultures of potato and tomato

The continuous replication of potato spindle tuber viroid (PSTV) in callus cultures from PSTV-infected wild-type potato (Solanum dem/ssum L.) and tomato (Lycopersicon peruvianum L. Mill) plants and in cell suspensions derived from potato protoplasts (Solanum tuberosum L.) inoculatedin vitro is described. The persistence of PSTV replication in these cell lines through at least 14 subculture passages, which corresponds to a continous replication over a period of more than one year, was demonstrated by infectivity assay and by polyacrylamide-gel electrophoresis of isolated nucleic acids. This continuous synthesis denovo of PSTV was substantiated by the incorporation of [³H]uridine and of [³²P]orthophosphate into viroid RNA.     

2,4-Dichlorophenoxyacetic Acid Inhibits the Outer Membrane NADH Dehydrogenase of Plant Mitochondria

The NADH dehydrogenase of potato (Solanum tuberosum) and mung bean (Phaseolus aureus) outer mitochondrial membranes is specifically inhibited by both 2,4-dichlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids but not by the natural auxin indole-3-acetic acid.     

An enriched preparation of basal-lateral plasma membranes from gastric glandular cells

Summary A procedure is described for the preparation of a membrane fraction enriched in basal-lateral plasma membranes from gastric mucosa. Gastric glands isolated from rabbit were employed as starting material, greatly reducing contamination from nonglandular cell types. The distribution of cellular components during the fractionation procedure was monitored with specific marker enzymes. (Na⁺+K⁺)-ATPase, ouabain-sensitive K⁺-stimulatedp-nitrophenyl-phosphatase and histamine-stimulated adenylate cyclase were used as markers for basal-lateral membranes. These three markers were similarly distributed during both differential and equilibrium density gradient centrifugation. The enriched membrane fraction contained more than 30% of the total initial activities of the three basal-lateral membrane markers which were purified better than 11-fold with respect to protein. (Na⁺+K⁺)-ATPase activity was resolved from the activities of acid phosphatase, pepsin, Mg²⁺-ATPase, cytochromec oxidase, NADPH-cytochromec reductase, glucose-6-phosphatase, (K⁺+H⁺)-ATPase, DNA and RNA.     

Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.Abbreviations LDMS laser desorption mass spectrometry - KDO 3-Deoxy-d-manno-octulosonic acid - Ts5 Salmonella typhimurium mutant Ts5 - PCP phenol/chloroform/petroleum ether - H₂F₂ hydrogen fluoride This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday     

High efficiency organogenesis and analysis of genetic stability of the regenerants in <Emphasis Type="Italic">Solanum melongena</Emphasis>

A novel protocol for plant regeneration from cotyledon explants of eggplant (Solanum melongena) reducing concentration of sucrose was established. The most efficient bud induction medium consisted of Murashige and Skoog (MS) medium supplemented with 2.0 mg dm⁻³ zeatin, 0.1 mg dm⁻³ indoleacetic acid and 10 g dm⁻³ sucrose. After 15 d, the shoot buds were fragmented and transferred to the shoot elongation MS supplemented with 1.0–2.0 mg dm⁻³ gibberellic acid and 4.0–8.0 mg dm⁻³ AgNO₃, which promoted shoots elongation. The genetic stability of the regenerated plants was analyzed by flow cytometry, RAPD and SSR molecular markers. The results indicated that almost no somaclonal variation was detected among the regenerants.     

The fatty acid constitution and ordering state of membranes in dominant temperature-sensitive lethal mutation and wild-typeDrosophila melanogaster larvae

The ordering state and changes in fatty acid composition of microsomal (MS) and mitochondrial (MC) membranes of two dominant temperature-sensitive (DTS) lethal mutations and the wild-type Oregon-R strain larvae ofDrosophila melanogaster have been studied at 18 and 29°C and after temperature-shift experiments. The membranes of wild-type larvae have a stable ordering state, with “S” values between 0.6 (18°C) and 0.5 (29°C) in both membranes which remained unchanged in shift experiments, although the ratios of saturated/unsaturated fatty acids were changed as expected. The stronglyDTS mutation1(2)10 ^DTS forms very rigid membranes at the restrictive temperature (29°C) which cannot be normalized after shift down, while shift up or development at the permissive temperature results in normal ordering state. This mutant is less able to adjust MS and MC fatty acid composition in response to the growth temperature than the wild type. The less temperature-sensitive1(2)2 ^DTS allele occupies an intermediate state between Oregon-R and1(2)10 ^DTS in both respects. We assume and the genetical data suggest that the DTS mutant gene product is in competition with the wild-type product, resulting in a membrane structure which is not able to accommodate to the restrictive temperature.     

Proton conductance through phospholipid bilayers: Water wires or weak acids?

The proton/hydroxide (H⁺/OH^–) permeability of phospholipid bilayer membranes at neutral pH is at least five orders of magnitude higher than the alkali or halide ion permeability, but the mechanism(s) of H⁺/OH^– transport are unknown. This review describes the characteristics of H⁺/OH^– permeability and conductance through several types of planar phospholipid bilayer membranes. At pH7, the H⁺/OH^– conductances (G _H/OH) range from 2–6 nS cm^–2, corresponding to net H⁺/OH^– permeabilities of (0.4–1.7)×10^–5 cm sec^–1. Inhibitors ofG _H/OH include serum albumin, phloretin, glycerol, and low pH. Enhancers ofG _H/OH include chlorodecane, fatty acids, gramicidin, and voltages >80 mV. Water permeability andG _H/OH are not correlated. The characteristics ofG _H/OH in fatty acid (weak acid) containing membranes are qualitatively similar to the controls in at least eight different respects. The characteristics ofG _H/OH in gramicidin (water wire) containing membranes are qualitatively different from the controls in at least four different respects. Thus, the simplest explanation for the data is thatG _H/OH in unmodified bilayers is due primarily to weakly acidic contaminants which act as proton carriers at physiological pH. However, at low pH or in the presence of inhibitors, a residualG _H/OH remains which may be due to water wires, hydrated defects, or other mechanisms.     

Effects of kinetin,paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light

Single-nodal cuttings of Solanum tuberosum (four cultivars) and Solanum chacoense were induced to produce in vitro microtubers on Murashige & Skoog (MS) medium supplemented with 8 g l^–1 sucrose and various concentrations of kinetin and paclobutrazol. The cultures were kept 10 days in darkness and then transferred to a 14 h daylength with 100 µE m^–2 sec^–1 light intensity at 21 °C. Kinetin (2.5 mg l^–1) had no significant influence on tuber formation. However, its addition together with paclobutrazol (0.001 mg l^–1) significantly enhanced tuberization. Paclobutrazol alone stimulated early tuber initiation and inhibited stem growth. Despite some genotype × treatment interactions, all genotypes (from very early to late and wild type) formed the maximum proportion of explants bearing microtubers on the media containing both plant growth regulators.     

Chemical Constituents of a Heat-dried Chinese Mushroom,Hohenbuehelia serotina

Several compounds were isolated from a Chinese mushroom, Huangmo, the heat-dried fruiting body of Hohenbuehelia serotina. They were identified as linoleic acid (I), hexadecanoic acid (II), β-sitosterol (IV), benzoic acid (V), D-mannitol (VI), sucrose (VII), and L-rhamnose (VIII). In addition, six acidic substances were identified. (Table I). Also, ethyl linoleate, hexadecanoic acid, and 9,12-octadecadienoic acid (Z-Z) ethyl esters, IV, V, and VI were identified for the first time from this mushroom.